Members of the family of BCL-2-related proteins serve as regulators of programmed cell death, or apoptosis (Cory, 1995 Annu. Rev. Immunol. 13, 513–543; Hockenbery, 1995 Nature 348, 334–336; Nunez et al., 1994 Nature 348, 334–336; Reed, 1994. J. Cell Biol. 124, 1–6; Akbar et al., 1993. Immunology Today 14, 526–532). Apoptosis has been shown to be involved in several immune processes, including intrathymic deletion of autoreactive cells, elimination of peripheral T cells during the response to viral and bacterial superantigens and lysis of target cells by cytotoxic T lymphocytes. There is increasing evidence that clonal expansion of antigen-specific T-cells is determined by the relative level of cellular proliferation and apoptosis following TCR ligation (Zinkemagel et al. 1993. Immunol. Rev. 131:199). However, the genetic mechanisms responsible for regulating these response phenotypes are not well understood.
The first gene to be identified which encoded a protein in this family, bcl-2, was cloned from the chromosomal breakpoint of t(14;18)-bearing B-cell lymphomas (Tsujimoto et al., 1984. Science 226:1097) and shown to inhibit cellular susceptibility to apoptosis (Cory, supra).
Several genes with homology to bcl-2 have subsequently been characterized, including the following: A1, which encodes an 80-amino acid protein that is rapidly induced in macrophages in response to GM-CSF or LPS (Lin et al., 1993. J. Immunol. 151, 1979–1988); MCL1, an early response gene in myeloid cell lines which undergo macrophage differentiation (Kozopas et al., 1993. Proc. Natl. Acad. Sci. USA 90, 3516–3520); and Bak, a BCL-2 homologue that may enhance apoptosis (Chittenden et al., 1995. Nature 374:733; Kiefer et al., 1995. Nature 374:736).
The bcl-x gene product, closely related to the BCL-2-related protein family, also protects cells from apoptosis. Analysis of mice deficient in BCL-x has suggested that its function is to support the viability of immature cells during development of the nervous and hematopoietic systems (Motoyama et al., 1995. Science 267, 1506–1510; Ma et al., 1995. Proc. Natl. Acad. Sci. USA 92, 4763–4767). Alternative splicing of human bcl-x may result in at least two distinct BCL-x mRNA species. The predominant protein product (233 amino acids) of the larger BCL-x mRNA, BCL-xL, inhibits cell death upon growth factor withdrawal (Boise et al., 1993. Cell 74, 597–608) and its transgenic expression alters thymocyte maturation leading to increased numbers of mature thymocytes (Chao et al., 1995. J. Exp. Med. 182, 821–828; Grillot et al., 1995. J. Exp. Med. 182, 1973–1983). After coligation of CD3 and CD28 in murine T-cells, enhanced BCL-xL expression may confer protection from apoptosis (Boise et al., 1995. Immunity 3, 87–98; Radvanyi et al., 1996. J. Immunol. 156, 1788–1798; Mueller et al., 1996. J. Immunol. 156, 1764–1771). The contribution of other isoforms of this gene to activation-induced death in T-cells is less well-defined (Gonzalez-Garcia et al., 1994. Development 120, 3033–3042; Fang et al., 1994 J. Immunol. 153, 4388–4398). A second human BCL-x isoform, BCL-xS, encodes a smaller protein of 170 amino acids which may enhance apoptosis, suggesting that different members of the BCL-x family may have opposing functions. Additional murine BCL-x isoforms, termed BCL-xβ and BCL-xΔTM, have been defined. The β isoform may inhibit apoptosis in neurons (Gonzalez-Garcia et al., 1995. Proc. Natl. Acad. Sci. U.S.A. 92, 4304–4308) and the ΔTM isoform may inhibit apoptosis in B-cells (Fang et al., supra).
Several proteins which interact with BCL-2 proteins have also been identified including bax, Nip1, Nip2, Nip 3, bad, and bag-1. These various BCL-2 binding proteins have different effects on apoptosis. For example, bak and bax function as inducers of apoptosis, whereas bag increases the resistance of cells to apoptosis (Farrow and Brown. 1996. Curr. Opin. Genetics and Devel. 6:45).
Despite the apparent importance of BCL-x in development and function of T-cells, none of the BCL-x isoforms described so far displays restricted expression with respect to this lineage; all four isoforms of BCL-x are ubiquitously expressed in a wide variety of tissues (Gonzalez-Garcia et al., 1994. Development 120, 3033–3042; Fang et al., supra). This may be because previous studies have isolated most of BCL-x isoforms (BCL-xL, BCL-xS and BCL-xΔTM) after screening cDNA libraries from tissues other than T-cells (Gonzalez-Garcia et al., 1994 supra; Fang et al., supra). The physiologic expression of these BCL-x isoforms is not sufficient to confer resistance to apoptosis following TCR ligation, since they are expressed equally well in apoptotic and non-apoptotic T-cell blasts. Moreover, overexpression of Bcl-xL does not affect thymocyte selection (Grillot et al. 1995. J. Exp. Med. 182:1973).